EP3812464A1 - Erhöhte resistenz gegen insekten und pflanzenpathogene ohne beeinträchtigung der samenproduktion - Google Patents

Erhöhte resistenz gegen insekten und pflanzenpathogene ohne beeinträchtigung der samenproduktion Download PDF

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EP3812464A1
EP3812464A1 EP20202431.1A EP20202431A EP3812464A1 EP 3812464 A1 EP3812464 A1 EP 3812464A1 EP 20202431 A EP20202431 A EP 20202431A EP 3812464 A1 EP3812464 A1 EP 3812464A1
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plant
endogenous
cdk8
plants
jaz
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Gregg A. Howe
Qiang Guo
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Michigan State University MSU
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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Definitions

  • Described herein are plants and methods providing improved defenses to increased resistance to pests and environmental stresses.
  • the plants and method involve jaz mutations to reduce JAZ repressors of defense (that can reduce plant growth) combined with CYCLIN-DEPENDENT KINASE 8 ( CDK8 ) mutations that restore growth of the jaz mutant plants without compromising the elevated pest defense.
  • Plants with reduced JAZ expression and/or reduced JAZ functioning have reduced growth, and a smaller stature.
  • combining loss of JAZ with loss of CDK8 functioning can lead to plants that exhibit good vegetative growth stature while simultaneously maintaining strong biotic stress resistance to insects and pathogens.
  • One example of a plant line with reduced JAZ functioning is the jazD plant line. Mutation of CDK8 in the jazD genetic background improved the reproductive output of jazD, achieving seed yields that were comparable to or even greater than wild type plants. Therefore, described herein is a useful strategy to promote strong pest and biotic stress resistance while improving seed production and growth.
  • the plants can have one or more loss of function mutations in at least one JAZ gene.
  • plants, and seeds described herein have endogenous jazD mutations that include mutations in the genes encoding JAZ1, JAZ2, JAZ3, JAZ4, JAZ5, JAZ6, JAZ7, JAZ9, JAZ10, and/or JAZ13 proteins.
  • Such mutations have reduced JAZ1, JAZ2, JAZ3, JAZ4, JAZ5, JAZ6, JAZ7, JAZ9, JAZ10, and/or JAZ13 activity.
  • JAZ1, JAZ2, JAZ3, JAZ4, JAZ5, JAZ6, JAZ7, JAZ9, JAZ10, and/or JAZ13 proteins is undetectable.
  • mutant cdk8 plant cells mutant cdk8 plants, and/or mutant cdk8 seeds the endogenous CDK8 proteins have reduced activity or their expression is undetectable.
  • endogenous JAZ8, JAZ11, and JAZ12 genes are not modified or mutated in the jaz cdk8 plant cells, plants and plant seeds.
  • endogenous JAZ8, JAZ11, and JAZ12 proteins can still be active in some cells and can be expressed in the mutant Jaz cdk8 plant cells, plants and/or plant seeds.
  • the plants or a plant grown from the seeds described herein have at least 5% less leaf damage from insect feeding than a wild type plant of the same species grown under the same conditions. In some cases, the plants or a plant grown from the seeds described herein have the same or at least about 10% more seed yield than a wild type plant of the same species grown under the same conditions.
  • the jazD plants by comparison to wild-type (WT) and jazQ plants, are highly resistant to both insect herbivores and necrotrophic pathogens but also exhibit reduced vegetative growth and reduced seed yield.
  • WT wild-type
  • jazQ plants are highly resistant to both insect herbivores and necrotrophic pathogens but also exhibit reduced vegetative growth and reduced seed yield.
  • CDK8 loss-of-function mutations when the jazD loss-of-function mutations are coupled with CDK8 loss-of-function mutations, plant growth is restored while the plants maintain strong biotic stress resistance to insects and pathogens.
  • mutation of CDK8 in the jazD genetic background seemed to improve the reproductive output of jazD, achieving seed yields that were comparable to or even greater than wild type plants.
  • jazD jazD
  • Plants and methods of making such plants are described herein that grow well and are resistant to environmental stresses such as drought and insects.
  • the plants have mutations that reduce or eliminate the expression or function of proteins that modulate jasmonic acid responses (e.g., JAZ genes/proteins). Plants with such mutations are referred to herein as jaz mutants or jaz plants.
  • jaz mutants or jaz plants Such reduction/elimination of jasmonic acid regulatory protein expression and/or function improves the resistance (compared to wild type plants) of jaz mutant plants to insects and biotic stress.
  • An additional mutation that reduces or eliminates the function of the cdk8 gene improves the growth of jazD mutant plants.
  • Plants with jazD mutations exhibit significantly improved resistance to insects and biotic stress, and when combined with loss-of-function cdk8 mutations, the plants grow reproduce well.
  • the jazD plants have loss-of-function mutations in ten JAZ genes: JAZ1, JAZ2, JAZ3, JAZ4, JAZ5, JAZ6, JAZ7, JAZ9, JAZ10, and JAZ13. Such jazD plants therefore have three remaining intact JAZ genes: JAZ8, JAZ11, and JAZ12.
  • plants with jazD mutations have transcription and/or translation of JAZ1, JAZ2, JAZ3, JAZ4, JAZ5, JAZ6, JAZ7, JAZ9, JAZ10, and JAZ13 reduced by at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% compared to wild type plant cells, plants, and seeds of the same species (that do not have the jazD ) .
  • plants with jazD mutations have transcription and/or translation of JAZ1, JAZ2, JAZ3, JAZ4, JAZ5, JAZ6, JAZ7, JAZ9, JAZ10, and JAZ13 reduced by at least 100%.
  • the jazD mutations are combined with loss-of-function cdk8 mutations.
  • plants with loss-of-function cdk8 mutations have transcription and/or translation of CDK8 reduced by at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% compared to wild type plant cells, plants, and seeds of the same species (that do not have the cdk8 loss-of-function mutation).
  • plants with cdk8 mutations have transcription and/or translation of CDK8 proteins reduced by at least 100%.
  • Non-limiting examples of methods of introducing a modification into the genome of a plant cell can include microinjection, viral delivery, recombinase technologies, homologous recombination, TALENS, CRISPR, and/or ZFN, see, e.g. Clark and Whitelaw Nature Reviews Genetics 4:825-833 (2003 ).
  • nucleases such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and/or meganucleases can be employed with guide nucleic acid that allows the nuclease to target the genomic JAZ and CDK8 site(s).
  • ZFNs zinc finger nucleases
  • TALENs transcription activator like effector nucleases
  • meganucleases can be employed with guide nucleic acid that allows the nuclease to target the genomic JAZ and CDK8 site(s).
  • a targeting vector can be used to introduce a deletion or modification of the genomic JAZ and CDK8 chromosomal sites.
  • a "targeting vector” is a vector generally has a 5' flanking region and a 3' flanking region homologous to segments of the gene of interest.
  • the 5' flanking region and a 3' flanking region can surround a DNA sequence comprising a modification and/or a foreign DNA sequence to be inserted into the gene.
  • the genomic JAZ and CDK8 site(s) can be disrupted by insertion of T-DNA.
  • the foreign DNA to be inserted may encode a selectable marker, such as an antibiotics resistance gene.
  • selectable markers examples include chloramphenicol resistance, gentamycin resistance, kanamycin resistance, spectinomycin resistance ( SpecR ), neomycin resistance gene (NEO) and hygromycin ⁇ -phosphotransferase markers (genes).
  • the 5' flanking region and the 3' flanking region can be homologous to regions within the gene, or such flanking regions can flank the coding region of gene to be deleted, mutated, or replaced with the unrelated DNA sequence.
  • the targeting vector does not comprise a selectable marker.
  • DNA comprising the targeting vector and the native gene of interest are contacted under conditions that favor homologous recombination (e.g., by transforming plant cell(s) with the targeting vector).
  • a typical targeting vector contains nucleic acid fragments of not less than about 0.1 kb nor more than about 10.0 kb from both the 5' and the 3' ends of the genomic locus which encodes the gene to be modified (e.g. the genomic JAZ and/or CDK8 site(s)). These two fragments can be separated by an intervening fragment of nucleic acid that includes the modification to be introduced.
  • the resulting construct recombines homologously with the chromosome at this locus, it results in the introduction of the modification, e.g. an insertion, substitution, or a deletion of a portion of the genomic JAZ and/or CDK8 site(s).
  • a Cas9/ CRISPR system can be used to create a modification in genomic JAZ and/or CDK8 site(s).
  • Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are useful for, e.g. RNA-programmable genome editing (see e.g., Marraffini & Sontheimer. Nature Reviews Genetics 11: 181-190 (2010 ); Sorek et al. Nature Reviews Microbiology 2008 6: 181-6 ; Karginov and Hannon. Mol Cell 2010 1 :7-19 ; Hale et al. Mol Cell 2010:45:292-302 ; Jinek et al.
  • a CRISPR guide RNA can be used that can target a Cas enzyme to the desired location in the genome, where it generates a double strand break. This technique is available in the art and described, e.g. at Mali et al. Science 2013 339:823-6 , and kits for the design and use of CRISPR-mediated genome editing are commercially available, e.g. the PRECISION X CAS9 SMART NUCLEASETM System (Cat No. CAS900A-1) from System Biosciences, Mountain View, CA.
  • cre-lox recombination system of bacteriophage P1, described by Abremski et al. 1983. Cell 32:1301 (1983 ), Sternberg et al., Cold Spring Harbor Symposia on Quantitative Biology, Vol. XLV 297 (1981 ) and others, can be used to promote recombination and alteration of the genomic JAZ and/or CDK8 site(s).
  • the cre-lox system utilizes the cre recombinase isolated from bacteriophage P1 in conjunction with the DNA sequences (termed lox sites) it recognizes. This recombination system has been effective for achieving recombination in plant cells ( U.S. Pat. No.
  • the plant cells, plants, and plant seeds can have genomic mutations that alter one or more amino acids in the encoded JAZ and/or CDK8 proteins.
  • plant cells, plants, and seeds can be modified so that at least one amino acid of a JAZ and/or CDK8 polypeptide is deleted or mutated to reduce the function of JAZ and/or CDK8 proteins.
  • a conserved amino acid or a conserved domain of the JAZ and/or CDK8 polypeptide is modified.
  • a conserved amino acid or several amino acids in a conserved domain of the JAZ and/or CDK8 polypeptide can be modified to change the physical and/or chemical properties of the conserved amino acid(s).
  • the amino acid(s) can be deleted or replaced by amino acid(s) of another class, where the classes are identified in the following Table 1.
  • Table 1 Classification Genetically Encoded Genetically Non-Encoded Hydrophobic Aromatic F, Y, W Phg, Nal, Thi, Tic, Phe(4-Cl), Phe(2-F), Phe(3-F), Phe(4-F), Pyridyl Ala, Benzothienyl Ala Apolar M, G, P Aliphatic A, V, L, I t-BuA, t-BuG, MeIle, Nle, MeVal, Cha, bAla, MeGly, Aib Hydrophilic Acidic D, E Basic H, K, R Dpr, Orn, hArg, Phe(p-NH 2 ), DBU, A 2 BU Polar Q, N, S, T, Y Cit, AcLys, MSO, hSer Cy
  • modified JAZ and/or CDK8 proteins can have any naturally occurring amino acid within the protein replaced with any of the amino acids listed in Tables 1 or 2.
  • jaz and/or cdk8 mutations are introduced by insertion of foreign DNA into the gene of interest.
  • this can involve the use of either transposable elements (see, e.g., Parinov et al., Plant Cell 11, 2263-2270 (1999 )) or T-DNA.
  • the foreign DNA not only disrupts the expression of the gene into which it is inserted but also acts as a marker for subsequent identification of the mutation. Because some plant introns are small, and because there can be very little intergenic material in plant chromosomes, the insertion of a piece of T-DNA on the order of 5 to 25 kb in length generally produces a dramatic disruption of gene function. If a large enough population of T-DNA-transformed lines is available, one has a very good chance of finding a plant carrying a T-DNA insert within any gene of interest.
  • Mutations that are homozygous lethal can be maintained in the population in the form of heterozygous plants.
  • Table 3 illustrates jaz mutations that can be combined to generate jazD mutant strains.
  • Mutants used for construction of jazD and jazU Mutant Original name Source Accession Mutagen Resistance 1 jaz1-2 SM_3.22668 JIC SM Col-0 dSpm transposon Basta (confirmed) jaz2-3 RIKEN_13 -5433-1 RIKEN No-0 Ds transposon Hygromycin (confirmed) jaz3-4 GK-097F09 GABI Kat Col-0 T-DNA (pAC161) Sulfadiazine (confirmed) jaz4-1 SALK_141628 SALK Col-0 T-DNA (pROK2) Kanamycin (silenced) jaz5-1 SALK_053775 SALK Col-0 T-DNA (pROK2) Kanamycin (confirmed) jaz6-4 CSHL_ET30 CSHL Ler Ds transposon (Enhancer trap GUS) Kanamycin (confirmed) jaz7
  • JAZ transcriptional repressor genes can be modified to improve insect and biotic resistance in plants.
  • the JAZ transcriptional repressor genes can encode JAZ1, JAZ2, JAZ3, JAZ4, JAZ5, JAZ6, JAZ7, JAZ9, JAZ10, JAZ13, and/or related proteins. Reduction or deletion of genes that encode JAZ1, JAZ2, JAZ3, JAZ4, JAZ5, JAZ6, JAZ7, JAZ9, JAZ10, JAZ13, and/or related proteins can provide insect and biotic resistance to plants.
  • JAZ1 proteins are repressors of the jasmonic acid signaling pathway.
  • JAZ1 Arabidopsis thaliana jasmonate-zim-domain protein 1
  • JAZ1 Arabidopsis thaliana jasmonate-zim-domain protein 1
  • JAZ2 is a coronatine (COR) and jasmonate isoleucine (JA-Ile) co-receptor, and is constitutively expressed in guard cells and modulates stomatal dynamics during bacterial invasion. It is expressed in cotyledons, hypocotyls, roots, sepals, petal vascular tissue and stigmas of developing flowers. JAZ2 is also expressed in stamen filaments after jasmonic acid treatment.
  • JAZ2 Arabidopsis thaliana jasmonate-zim-domain protein 2 (JAZ2) protein sequence is shown below (SEQ ID NO:3).
  • JAZ2 The Arabidopsis thaliana jasmonate-zim-domain 2 (JAZ2) gene resides on chromosome 1.
  • a cDNA encoding the protein with SEQ ID NO:3 is shown below as SEQ ID NO:4.
  • JAZ3 is also a repressor of jasmonate responses, and it is targeted by the SCF(COI1) complex for proteasome degradation in response to jasmonate.
  • SCF(COI1) the SCF(COI1) complex for proteasome degradation in response to jasmonate.
  • JAZ3 Arabidopsis thaliana jasmonate-zim-domain protein 3 (JAZ3) protein sequence is shown below (SEQ ID NO:5).
  • JAZ3 Arabidopsis thaliana jasmonate-zim-domain protein 3
  • JAZ4 is also a repressor of jasmonate responses.
  • JAZ4 Arabidopsis thaliana jasmonate-zim-domain protein 4 (JAZ4) protein sequence is shown below (SEQ ID NO:7).
  • JAZ4 Arabidopsis thaliana jasmonate-zim-domain protein 4
  • JAZ5 is also a repressor of jasmonate responses.
  • JAZ5 Arabidopsis thaliana jasmonate-zim-domain protein 5 (JAZ5) protein sequence is shown below (SEQ ID NO:9).
  • JAZ5 Arabidopsis thaliana jasmonate-zim-domain protein 5
  • JAZ6 is also a repressor of jasmonate responses.
  • JAZ6 Arabidopsis thaliana jasmonate-zim-domain protein 6 (JAZ6) protein sequence is shown below (SEQ ID NO:11).
  • JAZ6 Arabidopsis thaliana jasmonate-zim-domain protein 6
  • JAZ7 is also a repressor of jasmonate responses.
  • JAZ7 Arabidopsis thaliana jasmonate-zim-domain protein 7 (JAZ7) protein sequence is shown below (SEQ ID NO:13).
  • JAZ7 Arabidopsis thaliana jasmonate-zim-domain protein 7
  • JAZ9 is also a repressor of jasmonate responses.
  • JAZ9 Arabidopsis thaliana jasmonate-zim-domain protein 9 (JAZ9) protein sequence is shown below (SEQ ID NO: 15).
  • JAZ9 Arabidopsis thaliana jasmonate-zim-domain protein 9
  • JAZ10 is also a repressor of jasmonate responses.
  • JAZ10 Arabidopsis thaliana jasmonate-zim-domain protein 10 (JAZ10) protein sequence is shown below (SEQ ID NO:17).
  • JAZ10 Arabidopsis thaliana jasmonate-zim-domain protein 10
  • JAZ13 is also a repressor of jasmonate responses.
  • JAZ13 Arabidopsis thaliana jasmonate-zim-domain protein 13 (JAZ13) protein sequence is shown below (SEQ ID NO:19).
  • the Arabidopsis thaliana Jaz13 gene encoding the JAZ13 protein with SEQ ID NO:19 is located on chromosome 3, and a cDNA encoding the SEQ ID NO:19 is shown below as SEQ ID NO:20.
  • Chromosomal sequences that encode repressors of jasmonic acid responses from many plant types and species can be modified to reduce or eliminate the expression and/or function of the encoded protein.
  • chromosomal sequences encoding jasmonic acid repressor genes from agriculturally important plants such as alfalfa (e.g., forage legume alfalfa), algae, avocado, barley, broccoli, Brussels sprouts, cabbage, canola, cassava, cauliflower, cole vegetables, collards, corn, crucifers, grain legumes, grasses (e.g., forage grasses), jatropa, kale, kohlrabi, maize, miscanthus, mustards, nut sedge, oats, oil firewood trees, oilseeds, potato, radish, rape, rapeseed, rice, rutabaga, sorghum, soybean, sugar beets, sugarcane, sunflower, switchgrass, tobacco, tomato, turnips, and/or
  • more than one genetic or chromosomal segment encoding a jasmonic acid regulatory protein can be modified to reduce or eliminate the expression and/or function of the encoded protein(s).
  • more than two genes or chromosomal segments encoding jasmonic acid regulatory proteins can be modified to reduce or eliminate the expression and/or function of the encoded proteins.
  • more than three genes or chromosomal segments encoding jasmonic acid regulatory proteins can be modified to reduce or eliminate the expression and/or function of the encoded proteins.
  • more than four genes or chromosomal segments encoding jasmonic acid regulatory proteins can be modified to reduce or eliminate the expression and/or function of the encoded proteins.
  • JAZ-related proteins and nucleic acids that can be modified to reduce or eliminate the expression and/or function thereof, and thereby generate plants with improved resistance to insects.
  • TIFY 10A-like (NCBI accession no. XP_009117562.1; GI:685367109; SEQ ID NO:21) has significant sequence identity to the Arabidopsis thaliana JAZ1 protein with SEQ ID NO:1, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below the sequence comparison. 73.0% identity in 211 residues overlap; Score: 634.0; Gap frequency: 11.4%
  • This JAZ-related Brassica rapa protein, called TIFY 10A-like (NCBI accession no. XP_009117562.1; GI:685367109), has the following sequence (SEQ ID NO:21).
  • a cDNA encoding the SEQ ID NO:21 protein is available as NCBI accession number XM_009119314.1 (GI:685367108), and a chromosomal segment encoding the SEQ ID NO:21 protein is available as NCBI accession number AENI01008623.1 (GI:339949964).
  • Brassica oleracea protein also referred to as protein TIFY 10A-like (NCBI accession no. XP_013583936.1; GI:922487335; SEQ ID NO:22), has significant sequence identity to the Arabidopsis thaliana JAZ1 protein with SEQ ID NO:1, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below the sequence comparison. 72.9% identity in 192 residues overlap; Score: 633.0; Gap frequency: 2.6%
  • This JAZ-related Brassica oleracea protein referred to as protein TIFY 10A-like has the following sequence (SEQ ID NO:22).
  • a cDNA encoding the SEQ ID NO:22 protein is available as NCBI accession number XM_013728482.1 (GI:922487334), and a chromosomal segment encoding the SEQ ID NO:22 protein is available as NCBI accession number NC_027752.1 (GI:919506312).
  • LOC100276383 An uncharacterized Zea mays protein referred to as LOC100276383 (NCBI accession no. NP_001308779.1 (GI:1013071036) has significant sequence identity to the Arabidopsis thaliana JAZ1 protein with SEQ ID NO:1, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below the sequence comparison. 39.0% identity in 123 residues overlap; Score: 201.0; Gap frequency: 0.8%
  • LOC100276383 NCBI accession no. NP_001308779.1 (GI:1013071036) has the following sequence (SEQ ID NO:23).
  • a cDNA encoding the SEQ ID NO:23 protein is available as NCBI accession number NM_001321850.1 (GI:1013071035), and a chromosomal segment encoding the SEQ ID NO:23 protein is on Zea mays chromosome 7 at NC_024465.1 (165496371..165497455), sequence available as NCBI accession number NC_024465.1 (GI:662248746).
  • a Glycine max protein referred to as protein TIFY 10A-like (NCBI accession no. NP_001276307.1 (GI:574584782)) has significant sequence identity to the Arabidopsis thaliana JAZ1 protein with SEQ ID NO:1, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below the sequence comparison. 45.5% identity in 145 residues overlap; Score: 271.0; Gap frequency: 4.8%
  • This JAZ-related Glycine max protein referred to as protein TIFY 10A-like (NCBI accession no. NP_001276307.1 (GI:574584782) has the following sequence (SEQ ID NO:24).
  • a cDNA encoding the SEQ ID NO:24 protein is available as NCBI accession number NM_001289378.1 (GI:574584781), and a chromosomal segment encoding the SEQ ID NO:24 protein is on Glycine max chromosome 13 at NC_016100.2 (22541885..22544240), sequence available as NCBI accession number NC_016100.2 (GI:952545303).
  • An Oryza sativa protein referred to as protein TIFY 10b (Japonica Group; NCBI accession no. XP_015647536.1 (GI:1002286463) has significant sequence identity to the Arabidopsis thaliana JAZ1 protein with SEQ ID NO:1, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below the sequence comparison. 38.5% identity in 156 residues overlap; Score: 213.0; Gap frequency: 4.5% This JAZ-related Oryza sativa protein referred to as protein TIFY 10b (Japonica Group; NCBI accession no.
  • XP_015647536.1 (GI: 1002286463) that has significant sequence identity to the Arabidopsis thaliana JAZ1 protein, has the following sequence (SEQ ID NO:25).
  • a cDNA encoding the SEQ ID NO:25 protein is available as NCBI accession number XM_015792050.1 (GI:1002286462), and a chromosomal segment encoding the SEQ ID NO:25 protein is on Oryza sativa chromosome 7 at NC_029262.1 (25347990..25350243), sequence available as NCBI accession number NC_029262.1 (GI:996703426).
  • An uncharacterized Zea mays protein with NCBI accession no. ACF88234.1 (SEQ ID NO:26) has significant sequence identity to the Arabidopsis thaliana JAZ2 protein with SEQ ID NO:3, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below the sequence comparison. 35.7% identity in 235 residues overlap; Score: 227.0; Gap frequency: 9.8%
  • This JAZ-related Zea mays protein with NCBI accession no. ACF88234.1 that has significant sequence identity to the Arabidopsis thaliana JAZ2 protein has the following sequence (SEQ ID NO:26).
  • This JAZ-related Zea mays protein with NCBI accession no. ACF88234.1 is encoded by a gene on chromosome 2 at NC_024460.2 (218018545..218021029) of the Zea mays genome.
  • Triticum aestivum (wheat) protein with NCBI accession no. SPT16989.1 has significant sequence identity to the Arabidopsis thaliana JAZ2 protein with SEQ ID NO:3, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below the sequence comparison. 44.8% identity in 116 residues overlap; Score: 201.0; Gap frequency: 5.2%
  • This JAZ-related Triticum aestivum (wheat) with NCBI accession no. SPT16989.1 that has significant sequence identity to the Arabidopsis thaliana JAZ2 protein has the following sequence (SEQ ID NO:27).
  • Glycine max (soybean) protein with NCBI accession no. XP_003542368.1 (SEQ ID NO:28) has significant sequence identity to the Arabidopsis thaliana JAZ2 protein with SEQ ID NO:3, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below the sequence comparison. 42.6% identity in 230 residues overlap; Score: 314.0; Gap frequency: 12.6%
  • This JAZ-related Glycine max protein with NCBI accession no. XP_003542368.1 that has significant sequence identity to the Arabidopsis thaliana JAZ2 protein has the following sequence (SEQ ID NO:28).
  • This JAZ-related Glycine max protein with NCBI accession no. XP_003542368.1 is encoded by a gene at NC_038249.1 (22541885..22544240) on chromosome 13 of the Glycine max genome.
  • LOC103647411 NCBI accession no. NP_001288506.1; SEQ ID NO:29
  • SEQ ID NO:5 Arabidopsis thaliana JAZ3 protein with SEQ ID NO:5
  • Domains of sequence homology are identified by asterisks below the sequence comparison. 36.6% identity in 161 residues overlap; Score: 165.0; Gap frequency: 6.8%
  • This JAZ-related Zea mays protein referred to as LOC103647411 (NCBI accession no. NP_001288506.1) has the following sequence (SEQ ID NO:29).
  • a cDNA encoding the SEQ ID NO:29 protein is available as NCBI accession number NM_001301577.1 and a chromosomal segment encoding the SEQ ID NO:29 protein is on chromosome 2 at NC_024460.2 (184842608..184845336, complement) of the Zea mays genome, sequence available as NCBI accession number NC_024460.1 (GI:662249846).
  • a Triticum aestivum jasmonate ZIM-domain transcriptional repressor protein with (NCBI accession no. QBQ83004.1; SEQ ID NO:30) has significant sequence identity to the Arabidopsis thaliana JAZ3 protein with SEQ ID NO:5, as illustrated by the sequence comparison shown below, where the two sequences have about 30% sequence identity. Domains of sequence homology are identified by asterisks below the sequence comparison.
  • Triticum aestivum jasmonate ZIM-domain transcriptional repressor protein with significant sequence identity to the Arabidopsis thaliana JAZ3 protein with SEQ ID NO:5 and NCBI accession no. QBQ83004.1 has the following sequence (SEQ ID NO:30).
  • a cDNA encoding the SEQ ID NO:30 Triticum aestivum jasmonate protein has the sequence provided as NCBI accession number MH063273.1.
  • a Glycine max protein referred to as protein TIFY 6B-like isoform X1 (NCBI accession no. XP_003534135.1 (GI:356531138; SEQ ID NO:31) has significant sequence identity to the Arabidopsis thaliana JAZ3 protein with SEQ ID NO:5, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below the sequence comparison. 38.9% identity in 378 residues overlap; Score: 417.0; Gap frequency: 8.5%
  • This JAZ-related Glycine max protein referred to as protein TIFY 6B-like isoform X1 (NCBI accession no.
  • XP_003534135.1 (GI:356531138) has the following sequence (SEQ ID NO:31).
  • a cDNA encoding the SEQ ID NO:31 protein is available as NCBI accession number XM_003534087.3 (GI:955341633), and a chromosomal segment encoding the SEQ ID NO:31 protein is on Glycine max chromosome 9 at NC_016096.2 (39883473..39889992), sequence available as NCBI accession number NC_016096.2 (GI:952545307).
  • An Oryza sativa protein referred to as protein TIFY 6b (NCBI accession no. XP_015612402.1 (GI:1002297967), SEQ ID NO:32) has significant sequence identity to the Arabidopsis thaliana JAZ3 protein with SEQ ID NO:5, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below the sequence comparison. 37.3% identity in 177 residues overlap; Score: 142.0; Gap frequency: 10.2%
  • This JAZ-related Oryza sativa protein, referred to as protein TIFY 6b (NCBI accession no. XP_015612402.1 (GI:1002297967), has the following sequence (SEQ ID NO:32).
  • a cDNA encoding the SEQ ID NO:32 protein is available as NCBI accession number XM_015756916.1 (GI:1002297966), and a chromosomal segment encoding the SEQ ID NO:32 protein is on Oryza sativa chromosome 9 at NC_029264.1 (14056084..14060320, complement), sequence available as NCBI accession number NC_029264.1 (GI:996703424).
  • LOC100273108 An uncharacterized Zea mays protein referred to as LOC100273108 (NCBI accession no. NP_001141029.1 (GI:226500626), SEQ ID NO:33) has significant sequence identity to the Arabidopsis thaliana JAZ4 protein with SEQ ID NO:7.
  • the Zea mays SEQ ID NO:33 protein has domains of 40 residues having 55% sequence identity from positions 138-178, and 26 residues having 77% sequence identity from positions 258-284 homology with the Arabidopsis thaliana JAZ4 protein.
  • This JAZ-related uncharacterized Zea mays protein referred to as LOC100273108 (NCBI accession no.
  • NP_001141029.1 (GI:226500626), has the following sequence (SEQ ID NO:33).
  • a cDNA encoding the SEQ ID NO:33 protein is available as NCBI accession number NM_001147557.1 (GI:226500625), and a chromosomal segment encoding the SEQ ID NO:33 protein is on Zea mays chromosome 7 at NC_024465.1 (108871356..108874213, complement), sequence available as NCBI accession number NC_024465.1 (GI:662248746).
  • a Glycine max protein referred to as protein TIFY 6B isoform X5 (NCBI accession number XP_006580448.1 (GI:571456655; SEQ ID NO:34), has significant sequence identity to the Arabidopsis thaliana JAZ4 protein with SEQ ID NO:7, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified with asterisks below the sequence comparison. 37.0% identity in 322 residues overlap; Score: 273.0; Gap frequency: 8.7%
  • This JAZ-related Glycine max protein referred to as protein TIFY 6B isoform X5 (NCBI accession number XP_006580448.1 (GI:571456655), has the following sequence (SEQ ID NO:34).
  • a cDNA encoding the SEQ ID NO:34 protein is available as NCBI accession number XM_006580385.2 (GI:955322108), and a chromosomal segment encoding the SEQ ID NO:34 protein is on Glycine max chromosome 5 at NC_016092.2 (41222014..41225906), sequence available as NCBI accession number NC_016092.2 (GI:952545311).
  • An Oryza sativa protein referred to as protein TIFY 6a isoform X2 (NCBI accession number XP_015651050.1 (GI:1002293416; SEQ ID NO:35), has significant sequence identity to the Arabidopsis thaliana JAZ4 protein with SEQ ID NO:7.
  • the Oryza sativa SEQ ID NO:35 protein has domains of 26 residues having 81% sequence identity from positions 258-284 of the Arabidopsis thaliana JAZ4 protein with SEQ ID NO:7, and 47 residues having 45% sequence identity from positions 138-185 of the Arabidopsis thaliana JAZ4 protein with SEQ ID NO:7.
  • This JAZ-related Oryza sativa protein referred to as protein TIFY 6a isoform X2 (NCBI accession number XP_015651050.1 (GI:1002293416), has the following sequence (SEQ ID NO:35).
  • a cDNA encoding the SEQ ID NO:35 protein is available as NCBI accession number XM_015795564.1 (GI:1002293415), and a chromosomal segment encoding the SEQ ID NO:35 protein is on Oryza sativa chromosome 8 at NC_029263.1 (20624989..20627964, complement), sequence available as NCBI accession number NC_029263.1 (GI:996703425).
  • a Triticum aestivum jasmonate ZIM-domain transcriptional repressor protein with NCBI accession no. ABK63978.1 (SEQ ID NO:36) has significant sequence identity to the Arabidopsis thaliana JAZ4 protein with SEQ ID NO:7.
  • the Triticum aestivum SEQ ID NO:36 protein has domains of 36 residues having 67% sequence identity from positions 139-175 of the Arabidopsis thaliana JAZ4 protein with SEQ ID NO:7 and 26 residues having 58% sequence identity from positions 258-284 of the Arabidopsis thaliana JAZ4 protein with SEQ ID NO:7.
  • This Triticum aestivum jasmonate ZIM-domain transcriptional repressor protein with NCBI accession no. ABK63978.1 has the following sequence (SEQ ID NO:36).
  • a Zea mays protein referred to as hypothetical protein Zm00014a_023069 protein with NCBI accession no. PWZ14661.1 (SEQ ID NO:37) has significant sequence identity to the Arabidopsis thaliana JAZ5 protein with SEQ ID NO:9, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 31.4% identity in 207 residues overlap; Score: 131.0; Gap frequency: 11.6%
  • This Zea mays protein referred to as hypothetical protein Zm00014a_023069 with NCBI accession no. PWZ14661.1 has the following sequence (SEQ ID NO:37).
  • a Glycine max protein referred to as a TIFY 10A protein with NCBI accession no. XP_003546514.1 (SEQ ID NO:38) has significant sequence identity to the Arabidopsis thaliana JAZ5 protein with SEQ ID NO:9, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 35.6% identity in 219 residues overlap; Score: 206.0; Gap frequency: 9.6%
  • This Glycine max protein referred to as a TIFY 10A protein with NCBI accession no. XP_003546514.1 has the following sequence (SEQ ID NO:38).
  • a cDNA encoding the SEQ ID NO:38 protein is available as NCBI accession no. XM_003546466.4 and a chromosomal segment encoding the SEQ ID NO:38 protein is on Glycine max chromosome 15 at NC_038251.l (17292772..17295396).
  • Triticum aestivum protein with NCBI accession no. SPT20417.1 (SEQ ID NO:39) has significant sequence identity to the Arabidopsis thaliana JAZ5 protein with SEQ ID NO:9, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 31.5% identity in 124 residues overlap; Score: 109.0; Gap frequency: 5.6%
  • This unnamed Triticum aestivum protein with NCBI accession no. SPT20417.1 has the following sequence (SEQ ID NO:39).
  • a Zea mays protein referred to as TIFY 10b with NCBI accession no. PWZ12604.1 has significant sequence identity to the Arabidopsis thaliana JAZ6 protein with SEQ ID NO:11, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 38.1% identity in 105 residues overlap; Score: 156.0; Gap frequency: 1.9%
  • This TIFY 10b Zea mays protein with NCBI accession no. PWZ12604.1 has the following sequence (SEQ ID NO:40).
  • a Glycine max protein referred to as TIFY 10a-like isoform X1 with NCBI accession no. XP_006587054.1 (SEQ ID NO:41) has significant sequence identity to the Arabidopsis thaliana JAZ6 protein with SEQ ID NO:11, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 33.0% identity in 227 residues overlap; Score: 233.0; Gap frequency: 6.2% Seq11 5 QAPEKSNFSQRCSLLSRYLKEKGSFGNINMGLARKSDLELAGKFDLKGQQNVIKKVETSE This Glycine max protein (TIFY 10a-like isoform X1) with NCBI accession no.
  • XP_006587054.1 has the following sequence (SEQ ID NO:41).
  • a chromosomal segment encoding the SEQ ID NO:41 protein is on Glycine max chromosome 9 at NC_038245.1 (7366501..7369207).
  • An Oryza sativa protein referred to as TIFY 10b with NCBI accession no. A2YNP2.1 (SEQ ID NO:42) has significant sequence identity to the Arabidopsis thaliana JAZ6 protein with SEQ ID NO:11, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 31.6% identity in 206 residues overlap; Score: 182.0; Gap frequency: 5.8% This Oryza sativa protein (TIFY 10b) with NCBI accession no. A2YNP2.1 has the following sequence (SEQ ID NO:42).
  • a Zea mays protein referred to as protein TIFY5 with NCBI accession no. PWZ15752.1 has significant sequence identity to the Arabidopsis thaliana JAZ7 protein with SEQ ID NO:13.
  • the Zea mays SEQ ID NO:43 protein has domains of 65 residues having 32% sequence identity from positions 26-91 of the Arabidopsis thaliana JAZ7 protein with SEQ ID NO:13 and 21 residues having 62% sequence identity from positions 122-143 of the Arabidopsis thaliana JAZ7 protein with SEQ ID NO:13.
  • This Zea mays protein referred to as protein TIFY 5 with NCBI accession no. PWZ15752.1 has the following sequence (SEQ ID NO:43).
  • a Glycine max protein referred to as protein TIFY5A with NCBI accession no. XP_003546080.1 (SEQ ID NO:44) has significant sequence identity to the Arabidopsis thaliana JAZ7 protein with SEQ ID NO:13, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 41.8% identity in 91 residues overlap; Score: 157.0; Gap frequency: 2.2%
  • This Glycine max protein referred to as protein TIFY5A with NCBI accession no. XP_003546080.1 has the following sequence (SEQ ID NO:44).
  • Triticum aestivum protein with NCBI accession no. SPT17867.1 (SEQ ID NO:45) has significant sequence identity to the Arabidopsis thaliana JAZ7 protein with SEQ ID NO:13.
  • the Triticum aestivum SEQ ID NO:45 protein has domains of 31 residues having 45% sequence identity from positions 61-92 of the Arabidopsis thaliana JAZ7 protein with SEQ ID NO:13 and 24 residues having 67% sequence identity from positions 122-146 of the Arabidopsis thaliana JAZ7 protein with SEQ ID NO:13.
  • This unnamed Triticum aestivum protein with NCBI accession no. SPT17867.1 has the following sequence (SEQ ID NO:45).
  • a Zea mays protein referred to as putative tify domain/CCT motif transcription factor family protein (NCBI accession no. DAA40037.1 (GI:414589466); SEQ ID NO:46) has significant sequence identity to the Arabidopsis thaliana JAZ9 protein with SEQ ID NO:15.
  • the Zea mays SEQ ID NO:46 protein has domains of 48 residues having 52% sequence identity from positions 218-266 of the Arabidopsis thaliana JAZ9 protein with SEQ ID NO:15, and 31 residues having 55% sequence identity from positions 119-150 of the Arabidopsis thaliana JAZ9 protein with SEQ ID NO:15.
  • This JAZ-related uncharacterized Zea mays protein referred to as putative tify domain/CCT motif transcription factor family protein (NCBI accession no. DAA40037.1 (GI:414589466)), has the following sequence (SEQ ID NO:46).
  • a chromosomal segment encoding the SEQ ID NO:46 protein is on Zea mays chromosome 2 at NC_024460.1 (180086924..180089758, complement), sequence available as NCBI accession number NC_024460.1 (GI:662249846).
  • a Glycine max protein referred to as protein TIFY 6A isoform X6 (NCBI accession no XP_006580449.1 (GI:571456657; SEQ ID NO:47) has significant sequence identity to the Arabidopsis thaliana JAZ9 protein with SEQ ID NO:15, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 39.8% identity in 176 residues overlap
  • This JAZ-related Glycine max protein referred to as protein TIFY 6A isoform X6 (NCBI accession no. XP_006580449.1 (GI:571456657)) has the following sequence (SEQ ID NO:47).
  • a cDNA encoding the SEQ ID NO:47 protein is available as NCBI accession number XM_006580386.2 (GI:955322109), and a chromosomal segment encoding the SEQ ID NO:47 protein is on Glycine max chromosome 5 at NC_016092.2 (41222014..41225906), sequence available as NCBI accession number NC_016092.2 (GI:952545311).
  • An unknown Oryza sativa protein with NCBI accession no. BAD28520.1 (GI:50251455; SEQ ID NO:48) has significant sequence identity to the Arabidopsis thaliana JAZ9 protein with SEQ ID NO:15.
  • the Oryza sativa SEQ ID NO:48 protein has domains of 66 residues having 41% sequence identity from positions 84-150 of the Arabidopsis thaliana JAZ9 protein with SEQ ID NO:15, and 41 residues having 56% sequence identity from positions 218-259 of the Arabidopsis thaliana JAZ9 protein with SEQ ID NO:15.
  • BAD28520.1 (GI:50251455) has the following sequence (SEQ ID NO:48).
  • a chromosomal segment encoding the SEQ ID NO:48 protein is on Oryza sativa chromosome 9 at NC_029264.1 (14056084..14060320, complement), sequence available as NCBI accession number NC_029264.1 (GI:996703424).
  • LOC100384222 NCBI accession no. NP_001182812.1 (GI:308044557); SEQ ID NO:49
  • LOC100384222 NCBI accession no. NP_001182812.1 (GI:308044557); SEQ ID NO:49
  • SEQ ID NO:49 has significant sequence identity to the Arabidopsis thaliana JAZ10 protein with SEQ ID NO:17, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 36.2% identity in 94 residues overlap; Score: 126.0; Gap frequency: 3.2%
  • This JAZ-related uncharacterized Zea mays protein referred to as LOC100384222 NCBI accession no. NP_001182812.1 (GI:308044557)
  • SEQ ID NO:49 has the following sequence (SEQ ID NO:49).
  • a cDNA encoding the SEQ ID NO:49 protein is available as NCBI accession number NM_001195883.1 (GI:308044556), and a chromosomal segment encoding the SEQ ID NO:49 protein is on Zea mays chromosome 7 at NC_024465.1 (121257106..121259180, complement), sequence available as NCBI accession number NC_024465.1 (GI:662248746).
  • LOC100306524 NCBI accession number NP_001236269.1 (GI:351723837; SEQ ID NO:50) has significant sequence identity to the Arabidopsis thaliana JAZ10 protein with SEQ ID NO:17, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 36.6% identity in 123 residues overlap; Score: 114.0; Gap frequency: 12.2%
  • LOC100306524 NCBI accession number NP_001236269.1 (GI:351723837) has the following sequence (SEQ ID NO:50).
  • a cDNA encoding the SEQ ID NO:50 protein is available as NCBI accession number NM_001249340.2 (GI:402766138), and a chromosomal segment encoding the SEQ ID NO:50 protein is on Glycine max chromosome 15 at NC_016102.2 (18552881..18556339), sequence available as NCBI accession number NC_016102.2 (GI:952545301).
  • An Oryza sativa protein referred to as protein TIFY 9 with NCBI accession no. XP_015634258.1 has significant sequence identity to the Arabidopsis thaliana JAZ10 protein with SEQ ID NO:17, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison. 40.0% identity in 110 residues overlap; Score: 119.0; Gap frequency: 13.6% 66.7% identity in 12 residues overlap; Score: 44.0; Gap frequency: 0.0% This JAZ-related Oryza sativa protein referred to as protein TIFY 9 with NCBI accession no.
  • XP_015634258.1 (GI:1002259863) has the following sequence (SEQ ID NO:51).
  • a cDNA encoding the SEQ ID NO:51 protein is available as NCBI accession number XM_015778772.1 (GI:1002259862), and a chromosomal segment encoding the SEQ ID NO:51 protein is on Oryza sativa chromosome 4 at NC_029259.1 (19492605..19497181), sequence available as NCBI accession number NC_029259.1 (GI:996703429).
  • LOC100217316 isoform X2 with NCBI accession no. XP_008667401.1 (SEQ ID NO:52) has significant sequence identity to the Arabidopsis thaliana JAZ13 protein with SEQ ID NO:19, as illustrated by the sequence comparison shown below. Domains of sequence homology are identified by asterisks below each sequence comparison.
  • a cDNA encoding the SEQ ID NO:52 protein is available as NCBI accession number XM_008669179.2, and a chromosomal segment encoding the SEQ ID NO:52 protein is on Zea mays chromosome 2 at NC_024460.2 (226688215..226698574).
  • Chromosomal sites encoding any of the conserved amino acids and conserved domains illustrated by the sequence comparisons shown above can be deleted or mutated to reduce the activity of the proteins described herein.
  • a wild type plant can express JAZ polypeptides or JAZ-related polypeptides with at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to any of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 17, 19, 21-51 or 52.
  • mutant jazD plant cells, plants, and/or seeds with improved insect and biotic stress resistance can express some JAZ and/or JAZ-related polypeptides such as the JAZ8, JAZ11, and JAZ12 proteins.
  • JAZ8, JAZ11, and JAZ12 genes are not modified or mutated in the jazD plant cells, plants, and seeds described herein.
  • jazD plant cells, plants, and/or seeds having reduced activity of JAZ1, JAZ2, JAZ3, JAZ4, JAZ5, JAZ6, JAZ7, JAZ9, JAZ10, and JAZ13 can have less than 99%, or less than 98%, or less than 95%, or less than 90%, or less than 85%, or less than 75%, or less than 60%, or less than 50%, or less than 40%, or less than 30%, or less than 20% sequence identity to any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 17, 19, 21-51 or 52.
  • the mutant JAZ and/or JAZ-related polypeptides can, for example, have mutations in at least one conserved amino acid position, or at least two conserved amino acid positions, or at least three conserved amino acid positions, or at least five conserved amino acid positions, or at least seven conserved amino acid positions, or at least eight conserved amino acid positions, or at least ten conserved amino acid positions, or at least fifteen amino acid positions, or at least twenty conserved amino acid positions, or at least twenty-five amino acid positions.
  • an entire conserved JAZ and/or JAZ-related domain or the entire endogenous JAZ and/or JAZ -related gene or chromosomal segment is deleted or mutated.
  • the conserved amino acids and/or domains are in some cases mutated by deletion or replacement with amino acids that have dissimilar physical and/or chemical properties. Examples of amino acids with different physical and/or chemical properties that can be employed are shown in Tables 1 and 2.
  • Cdk8 loss-of-function mutations of cdk8 can improve the pest resistance, poor growth, and poor reproduction of jazD mutant plants.
  • the Cdk8 gene is also named the CdkE1 or Hen3 gene in some species.
  • Arabidopsis thaliana CDK8 protein sequence is provided by accession no. AT5G63610.1, shown below as SEQ ID NO:53.
  • the wild type Arabidopsis thaliana CDK8 protein with SEQ ID NO:53 is encoded by a cDNA (At5G63610) with the following sequence (SEQ ID NO:54).
  • the CDK8 gene resides on chromosome 5 at 25463362 - 25465922 bp.
  • Chromosomal sequences that encode CDK8 proteins from many plant types and species can be modified to reduce or eliminate the expression and/or function of the encoded protein.
  • the Arabidopsis thaliana CDK8 gene can be mutated to generate a null allele such as the sjd56 mutant CDK8 allele, which has a C1684T mutation altering a glutamine reside to a stop codon in the encoded protein.
  • the sjd56 mutation is shown in the CDK8 SEQ ID NO:54 nucleic acid sequence below, now referred to as SEQ ID NO:55 and illustrating that the position of this mutation can vary by 20-30 nucleotides.
  • such sjd56 mutations of the CDK8 gene can improve plant pest resistance, growth, and seed production.
  • CDK8 genes from a variety of species can be modified (mutated) to improve their pest resistance, growth, and seed production.
  • chromosomal sequences encoding CDK8 genes from agriculturally important plants such as alfalfa (e.g., forage legume alfalfa), algae, avocado, barley, broccoli, Brussels sprouts, cabbage, canola, cassava, cauliflower, cole vegetables, collards, corn, crucifers, grain legumes, grasses (e.g., forage grasses), jatropa, kale, kohlrabi, maize, miscanthus, mustards, nut sedge, oats, oil firewood trees, oilseeds, potato, radish, rape, rapeseed, rice, rutabaga, sorghum, soybean, sugar beets, sugarcane, sunflower, switchgrass, tobacco, tomato, turnips, and/or wheat can be modified reduce or eliminate the expression and/or function of CDK8 proteins.
  • a wild type Zea mays CDK8 protein has NCBI accession number AQK66278.1, and the sequence shown below as SEQ ID NO:56.
  • the Zea mays CDK8 protein with SEQ ID NO:56 is encoded by the LOC100284562 gene on chromosome 5 at NC_024463.2 (46913511..46918664, complement).
  • a cDNA that encodes the SEQ ID NO:55 CDK8 protein is shown below as SEQ ID NO:57.
  • Another wild type Zea mays CDK8 protein has NCBI accession number PWZ24329.1, and the sequence shown below as SEQ ID NO:58.
  • a wild type Glycine max CDK8 protein has NCBI accession number XP_003532085.1, and the sequence shown below as SEQ ID NO:59.
  • the Glycine max CDK8 protein with SEQ ID NO:59 is encoded by the LOC100807993 gene on chromosome 8 at NC_038244.1 (211278..221643, complement).
  • a cDNA that encodes the SEQ ID NO:58 CDK8 protein is shown below as SEQ ID NO:60.
  • Another wild type Glycine max CDK8 protein has NCBI accession number XP_003525137.1, and the sequence shown below as SEQ ID NO:61.
  • the wild type Glycine max CDK8 protein with SEQ ID NO:61 is encoded by the LOC100794990 gene on chromosome 5 at NC_038241.1 (37955973..37967547, complement).
  • a cDNA that encodes the SEQ ID NO:61 CDK8 protein is shown below as SEQ ID NO:62.
  • a wild type Triticum aestivum CDK8 protein has NCBI accession number AAD10483.1, and the sequence shown below as SEQ ID NO:63.
  • the Triticum aestivum CDK8 protein with SEQ ID NO:63 is encoded by the cdc2TaA gene.
  • a cDNA that encodes the SEQ ID NO:62 CDK8 protein is shown below as SEQ ID NO:64.
  • a wild type Oryza sativa Japonica Group CDK8 protein has NCBI accession number XP_015614383.1, and the sequence shown below as SEQ ID NO:65.
  • the Oryza sativa CDK8 protein with SEQ ID NO:65 is encoded by the LOC4349519 gene on chromosome 10 at NC_029265.1 (23148732..23153285, complement).
  • a cDNA that encodes the SEQ ID NO:65 CDK8 protein is shown below as SEQ ID NO:66.
  • a wild type plant can have cdk8 nucleic acids or express CDK8 polypeptides or CDK8-related polypeptides with at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to any of SEQ ID NOs:53-66.
  • Plant cells from such wild type plants can be mutated, and mutant plants can be generated therefrom as described herein to provide modified jazD cdk8 plants and plant seed with improved plant growth and seed yields.
  • mutant cdk8 plant cells, plants, and/or seeds with increased jasmonic acid responses and improved insect resistance can express mutant CDK8 and/or CDK8-related polypeptides that have reduced activity. In some cases, detectable levels of CDK8 proteins are not expressed
  • Such cdk8 mutant plant cells and plant tissues have reduced CDK8 activity can cdk8 nucleic acids or cdk8 polypeptides that have less than 99%, or less than 98%, or less than 95%, or less than 90%, or less than 85%, or less than 75%, or less than 60%, or less than 50%, or less than 40%, or less than 30%, or less than 20% sequence identity to any of SEQ ID NOs: 53-66.
  • the mutant CDK8 and/or CDK8-related polypeptides can, for example, have mutations in at least one conserved amino acid position, or at least two conserved amino acid positions, or at least three conserved amino acid positions, or at least five conserved amino acid positions, or at least seven conserved amino acid positions, or at least eight conserved amino acid positions, or at least ten conserved amino acid positions, or at least fifteen amino acid positions, or at least twenty conserved amino acid positions, or at least twenty-five amino acid positions.
  • an entire conserved CDK8 and/or JAZ-related domain or the entire endogenous Cdk8 and/or Cdk8-related gene or chromosomal segment is deleted or mutated.
  • the conserved amino acids and/or domains are in some cases mutated by deletion or replacement with amino acids that have dissimilar physical and/or chemical properties. Examples of amino acids with different physical and/or chemical properties that can be employed are shown in Tables 1 and 2.
  • Mutations can be introduced into any of the wild type JAZ, JAZ-related, CDK8 or CDK8- related plant genomes by introducing targeting vectors, T-DNA, transposons, nucleic acids encoding TALENS, CRISPR, or ZFN nucleases, and combinations thereof into a recipient plant cell to create a transformed cell.
  • Cells from virtually any dicot or monocot species can be stably modified or transformed, and these cells can be regenerated into transgenic plants, through the application of the techniques disclosed herein.
  • the plant cells, plants, and seeds can therefore be monocotyledons or dicotyledons.
  • the cell(s) that undergo transformation may be in a suspension cell culture or may be in an intact plant part, such as an immature embryo, or in a specialized plant tissue, such as callus, such as Type I or Type II callus.
  • Transformation of the cells of the plant tissue source can be conducted by any one of a number of methods available to those of skill in the art. Examples include: Transformation by direct DNA transfer into plant cells by electroporation ( U.S. Patent No. 5,384,253 and U.S. Patent No. 5,472,869, Dekeyser et al. , The Plant Cell. 2:591 602 (1990 )); direct DNA transfer to plant cells by PEG precipitation ( Hayashimoto et al., Plant Physiol. 93:857 863 (1990 )); direct DNA transfer to plant cells by microprojectile bombardment ( McCabe et al., Bio/Technology. 6:923 926 (1988 ); Gordon Kamm et al., The Plant Cell.
  • One method for dicot transformation involves infection of plant cells with Agrobacterium tumefaciens using the leaf disk protocol ( Horsch et al., Science 227:1229 1231 (1985 ).
  • Monocots such as Zea mays can be transformed via microprojectile bombardment of embryogenic callus tissue or immature embryos, or by electroporation following partial enzymatic degradation of the cell wall with a pectinase containing enzyme ( U.S. Patent No. 5,384,253 ; and U.S. Patent No. 5,472,869 ).
  • embryogenic cell lines derived from immature Zea mays embryos can be transformed by accelerated particle treatment as described by Gordon Kamm et al. (The Plant Cell.
  • Excised immature embryos can also be used as the target for transformation prior to tissue culture induction, selection and regeneration as described in U.S. application Serial No. 08/112,245 and PCT publication WO 95/06128 .
  • methods for transformation of monocotyledonous plants utilizing Agrobacterium tumefaciens have been described by Hiei et al. (European Patent 0 604 662 , 1994 ) and Saito et al. (European Patent 0 672 752, 1995 ).
  • Methods such as microprojectile bombardment or electroporation can be carried out with "naked" DNA where the expression cassette may be simply carried, for example, on any E. coli derived plasmid cloning vector.
  • the system retain replication functions, but lack functions for disease induction.
  • tissue source for transformation will depend on the nature of the host plant and the transformation protocol.
  • Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like.
  • the tissue source is selected and transformed so that it retains the ability to regenerate whole, fertile plants following transformation, i.e., contains totipotent cells.
  • Type I or Type II embryonic maize callus and immature embryos are exemplary Zea mays tissue sources. Selection of tissue sources for transformation of monocots is described in detail in U.S. Application Serial No. 08/112,245 and PCT publication WO 95/06128 .
  • the transformation is carried out under conditions directed to the plant tissue of choice.
  • the plant cells or tissue are exposed to the DNA or RNA carrying the targeting vector and/or other nucleic acids for an effective period of time. This may range from a less than one second pulse of electricity for electroporation to a 2-3-day co-cultivation in the presence of plasmid bearing Agrobacterium cells. Buffers and media used will also vary with the plant tissue source and transformation protocol. Many transformation protocols employ a feeder layer of suspended culture cells (tobacco or Black Mexican Sweet corn, for example) on the surface of solid media plates, separated by a sterile filter paper disk from the plant cells or tissues being transformed.
  • suspended culture cells tobacco or Black Mexican Sweet corn, for example
  • Krzyzek et al. U.S. Patent No. 5,384,253
  • certain cell wall degrading enzymes such as pectin degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells.
  • recipient cells can be made more susceptible to transformation, by mechanical wounding.
  • friable tissues such as a suspension cell cultures, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly.
  • the cell walls of the preselected cells or organs can be partially degraded by exposing them to pectin degrading enzymes (pectinases or pectolyases) or mechanically wounding them in a controlled manner.
  • pectinases or pectolyases pectinases or pectolyases
  • Such cells would then be receptive to DNA uptake by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA.
  • microparticles may be coated with DNA and delivered into cells by a propelling force.
  • Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
  • DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment.
  • non-embryogenic cells were bombarded with intact cells of the bacteria E. coli or Agrobacterium tumefaciens containing plasmids with either the ⁇ -glucouronidase or bar gene engineered for expression in maize. Bacteria were inactivated by ethanol dehydration prior to bombardment. A low level of transient expression of the ⁇ -glucouronidase gene was observed 24-48 hours following DNA delivery.
  • stable transformants containing the bar gene can be recovered following bombardment with either E. coli or Agrobacterium tumefaciens cells. It is contemplated that particles may contain DNA rather than be coated with DNA. Hence it is proposed that particles may increase the level of DNA delivery but are not, in and of themselves, necessary to introduce DNA into plant cells.
  • An advantage of microprojectile bombardment in addition to being an effective means of reproducibly stably transforming monocots, is that the isolation of protoplasts ( Christou et al., PNAS. 84:3962 3966 (1987 )), the formation of partially degraded cells, or the susceptibility to Agrobacterium infection is not required.
  • An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with maize cells cultured in suspension ( Gordon Kamm et al., The Plant Cell. 2:603 618 (1990 )).
  • the screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectile aggregate and may contribute to a higher frequency of transformation, by reducing damage inflicted on the recipient cells by an aggregated projectile.
  • cells in suspension are preferably concentrated on filters or solid culture medium.
  • immature embryos or other target cells may be arranged on solid culture medium.
  • the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.
  • one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth here in one may obtain up to 1000 or more foci of cells transiently expressing a marker gene.
  • the number of cells in a focus which express the exogenous gene product 48 hours post bombardment often range from about 1 to 10 and average about 1 to 3.
  • bombardment transformation one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment can influence transformation frequency. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the path and velocity of either the macroprojectiles or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmid DNA.
  • TRFs trauma reduction factors
  • plants and/or plant cells that can be modified as described herein include alfalfa (e.g., forage legume alfalfa), algae, avocado, barley, broccoli, Brussels sprouts, cabbage, canola, cassava, cauliflower, cole vegetables, collards, corn, crucifers, grain legumes, grasses (e.g., forage grasses), jatropa, kale, kohlrabi, maize, miscanthus, mustards, nut sedge, oats, oil firewood trees, oilseeds, potato, radish, rape, rapeseed, rice, rutabaga, sorghum, soybean, sugar beets, sugarcane, sunflower, switchgrass, tobacco, tomato, turnips, and wheat.
  • alfalfa e.g., forage legume alfalfa
  • algae e.g., forage legume alfalfa
  • avocado e.g., broccoli, Brussels sprouts, cabbage, canola
  • the plant is a Brassicaceae or other Solanaceae species.
  • the plant or cell can be a maize plant or cell.
  • the plant is not a species of Arabidopsis, for example, in some embodiments, the plant is not Arabidopsis thaliana.
  • An exemplary embodiment of methods for identifying transformed cells involves exposing the bombarded cultures to a selective agent, such as a metabolic inhibitor, an antibiotic, herbicide or the like.
  • a selective agent such as a metabolic inhibitor, an antibiotic, herbicide or the like.
  • Cells which have been transformed and have stably integrated a marker gene conferring resistance to the selective agent used will grow and divide in culture. Sensitive cells will not be amenable to further culturing.
  • bombarded tissue is cultured for about 0-28 days on nonselective medium and subsequently transferred to medium containing from about 1-3 mg/l bialaphos or about 1-3 mM glyphosate, as appropriate. While ranges of about 1-3 mg/l bialaphos or about 1-3 mM glyphosate can be employed, it is proposed that ranges of at least about 0.1-50 mg/l bialaphos or at least about 0.1-50 mM glyphosate will find utility in the practice of the invention. Tissue can be placed on any porous, inert, solid or semi-solid support for bombardment, including but not limited to filters and solid culture medium. Bialaphos and glyphosate are provided as examples of agents suitable for selection of transformants, but the technique of this invention is not limited to them.
  • An example of a screenable marker trait is the red pigment produced under the control of the R-locus in maize. This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media.
  • the R-locus is useful for selection of transformants from bombarded immature embryos.
  • the introduction of the C1 and B genes will result in pigmented cells and/or tissues.
  • the enzyme luciferase is also useful as a screenable marker in the context of the present invention.
  • cells expressing luciferase emit light which can be detected on photographic or X-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification.
  • the photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells which are expressing luciferase and manipulate those in real time.
  • combinations of screenable and selectable markers may be useful for identification of transformed cells.
  • selection with a growth inhibiting compound, such as bialaphos or glyphosate at concentrations below those that cause 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase would allow one to recover transformants from cell or tissue types that are not amenable to selection alone.
  • embryogenic Type II callus of Zea mays L. can be selected with sub-lethal levels of bialaphos. Slowly growing tissue was subsequently screened for expression of the luciferase gene and transformants can be identified.
  • Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, are cultured in media that supports regeneration of plants.
  • a growth regulator that can be used for such purposes is dicamba or 2,4-D.
  • other growth regulators may be employed, including NAA, NAA + 2,4-D or perhaps even picloram.
  • Media improvement in these and like ways can facilitate the growth of cells at specific developmental stages. Tissue can be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, at least two weeks, then transferred to media conducive to maturation of embryoids. Cultures are typically transferred every two weeks on this medium. Shoot development signals the time to transfer to medium lacking growth regulators.
  • the transformed cells identified by selection or screening and cultured in an appropriate medium that supports regeneration, can then be allowed to mature into plants.
  • Developing plantlets are transferred to soilless plant growth mix, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, about 600 ppm CO 2 , and at about 25-250 microeinsteins/sec ⁇ m 2 of light.
  • Plants can be matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue.
  • cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant ConTM.
  • Regenerating plants can be grown at about 19 °C to 28 °C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.
  • Mature plants are then obtained from cell lines that are known to have the mutations.
  • the regenerated plants are self-pollinated.
  • pollen obtained from the regenerated plants can be crossed to seed grown plants of agronomically important inbred lines.
  • pollen from plants of these inbred lines is used to pollinate regenerated plants.
  • the trait is genetically characterized by evaluating the segregation of the trait in first and later generation progeny. The heritability and expression in plants of traits selected in tissue culture are of particular importance if the traits are to be commercially useful.
  • Regenerated plants can be repeatedly crossed to inbred plants in order to introgress the mutations into the genome of the inbred plants. This process is referred to as backcross conversion.
  • backcross conversion When a sufficient number of crosses to the recurrent inbred parent have been completed in order to produce a product of the backcross conversion process that is substantially isogenic with the recurrent inbred parent except for the presence of the introduced, Jaz or Cdk8 mutations, the plant is self-pollinated at least once in order to produce a homozygous backcross converted inbred containing the mutations. Progeny of these plants are true breeding.
  • seed from transformed mutant plant lines regenerated from transformed tissue cultures is grown in the field and self-pollinated to generate true breeding plants.
  • Transgenic plant and/or seed tissue can be analyzed using standard methods such as SDS polyacrylamide gel electrophoresis, liquid chromatography (e.g., HPLC) or other means of detecting a mutation.
  • transgenic plant with a mutant sequence and having improved growth and insect resistance seeds from such plants can be used to develop true breeding plants.
  • the true breeding plants are used to develop a line of plants with an increase insect resistance relative to wild type, and acceptable growth characteristics while still maintaining other desirable functional agronomic traits. Adding the mutation to other plants can be accomplished by back-crossing with this trait and with plants that do not exhibit this trait and studying the pattern of inheritance in segregating generations. Those plants expressing the target trait (insect resistance, good growth) in a dominant fashion are preferably selected.
  • Back-crossing is carried out by crossing the original fertile transgenic plants with a plant from an inbred line exhibiting desirable functional agronomic characteristics while not necessarily expressing the trait of an increased insect resistance and good plant growth.
  • the resulting progeny are then crossed back to the parent that expresses the increased insect resistance and good plant growth.
  • the progeny from this cross will also segregate so that some of the progeny carry the trait and some do not.
  • This back-crossing is repeated until an inbred line with the desirable functional agronomic traits, and with expression of the trait involving an increase in insect resistance and good plant growth. Such insect resistance and good plant growth can be expressed in a dominant fashion.
  • the new transgenic plants can also be evaluated for a battery of functional agronomic characteristics such as growth, lodging, kernel hardness, yield, resistance to disease and insect pests, drought resistance, and/or herbicide resistance.
  • Plants that may be improved by these methods include but are not limited to agricultural plants of all types, oil and/or starch plants (canola, potatoes, lupins, sunflower and cottonseed), forage plants (alfalfa, clover and fescue), grains (maize, wheat, barley, oats, rice, sorghum, millet and rye), grasses (switchgrass, prairie grass, wheat grass, sudangrass, sorghum, straw-producing plants), softwood, hardwood and other woody plants (e.g., those used for paper production such as poplar species, pine species, and eucalyptus).
  • the plant is a gymnosperm.
  • plants useful for pulp and paper production include most pine species such as loblolly pine, Jack pine, Southern pine, Radiata pine, spruce, Douglas fir and others. Hardwoods that can be modified as described herein include aspen, poplar, eucalyptus, and others. Plants useful for making biofuels and ethanol include corn, grasses (e.g., miscanthus, switchgrass, and the like), as well as trees such as poplar, aspen, willow, and the like. Plants useful for generating dairy forage include legumes such as alfalfa, as well as forage grasses such as bromegrass, and bluestem.
  • assays include, for example, molecular biological assays available to those of skill in the art, such as Southern and Northern blotting and PCR; biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf, seed or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
  • molecular biological assays available to those of skill in the art, such as Southern and Northern blotting and PCR
  • biochemical assays such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function
  • plant part assays such as leaf, seed or root assays
  • analyzing the phenotype of the whole regenerated plant include, for example, molecular biological assays available to those of skill in the art, such as Southern and Northern
  • RNA may only be expressed in particular cells or tissue types and so RNA for analysis can be obtained from those tissues.
  • PCR techniques may also be used for detection and quantification of RNA produced from the introduced Jaz or Cdk8 mutants.
  • PCR also be used to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then this DNA can be amplified through the use of conventional PCR techniques.
  • RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence of some mutations can be detected by Northern blotting. The presence or absence of an RNA species (e.g., a Jaz or cdk8 RNA) can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and also demonstrate the presence or absence of an RNA species.
  • an RNA species e.g., a Jaz or cdk8 RNA
  • Southern blotting and PCR may be used to detect the presence of Jaz, and/or cdk8 mutations or the presence of a PIF4 expression cassette, they do not provide information as to whether the preselected DNA segment is being expressed.
  • Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins.
  • Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange, liquid chromatography or gel exclusion chromatography.
  • electrophoretic procedures such as native or denaturing gel electrophoresis or isoelectric focusing
  • chromatographic techniques such as ion exchange, liquid chromatography or gel exclusion chromatography.
  • the unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products, or the absence thereof, that have been separated by electrophoretic techniques.
  • Additional techniques may be employed to absolutely confirm the identity of a mutation such as evaluation by screening for reduced transcription (or no transcription) of Jaz, and/or cdk8 mRNAs, or by amino acid sequencing following purification.
  • the Examples of this application also provide assay procedures for detecting and quantifying insect resistance and plant growth. Other procedures may be additionally used.
  • the expression of a gene product can also be determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the insect resistance, growth characteristics, or other physiological properties of the plant. Expression of selected DNA segments encoding different amino acids or having different sequences and may be detected by amino acid analysis or sequencing.
  • the jazD cdk8 plants and seeds described herein can also be identified and characterized phenotypically.
  • the jazD cdk8 plant's vegetative weight or vegetative weight of a jazD cdk8 plant grown from jazD cdk8 plant seeds is within at least about 40%, or at least about 50%, or within at least 60%, or at least about 70% of the average vegetative weight of a wild type plant grown for the same time and under the same conditions as a wild type plant.
  • jazD cdk8 plants or plants grown from jazD cdk8 plant seeds have a seed yield that is at least 10%, or at least 20%, or at least 30%, or at least 40% greater than the average seed yield of wild type plants.
  • the jazD cdk8 plants or plants grown from jazD cdk8 plant seeds have at least 5% less, 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less, or 100% less leaf damage from insect feeding than average insect feeding of a wild type plant of the same species grown for the same time under the same conditions.
  • the jazD cdk8 plants or plants grown from jazD cdk8 plant seeds have at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% fewer insects or insect larvae than an average number of insects or insect larvae of wild type plants of the same species grown for the same time under the same conditions.
  • loss of function mutations of Jaz and cdk8 genes can improve plant resistance to insects. Plants with such mutations can produce a variety of compounds that can repel, metabolically undermine, or otherwise discourage insects and/or insect larvae from infesting plant tissues. Such compounds are referred to as defense compounds. In some cases, the defense compounds are aliphatic glucosinolates. Examples of defense compounds include:
  • Mutation of jaz and/or cdk8 genes in plants can lead to increased synthesis of at least one defense compound, at least two defense compounds, at least three defense compounds, at least four defense compound, at least five defense compounds, at least six defense compounds, at least seven defense compound, at least eight defense compounds, or at least nine defense compounds.
  • the defense compounds can be produced by a variety of plant tissues.
  • plant tissues where the defense compounds can be made include leaves, stems, seeds, or a combination thereof.
  • plant leaves can have increased content of a variety of defense compounds in plants with loss of function JazD cdk8 genes, as illustrated in FIG. 12 .
  • the defense compounds can be at least 2%, at least 3%, at least 4%, at least 5%, at least 7%, at least 10%, at least 13%, at least 15%, at least 17%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90%, or at least 100% greater levels in plants with loss of function Jaz mutations, loss of function cdk8 mutations, or a combination thereof, than in unmodified parental or wild type plants.
  • heterologous when used in reference to a nucleic acid or protein refers to a nucleic acid or protein that has been manipulated in some way.
  • a heterologous nucleic acid includes a nucleic acid from one species introduced into another species.
  • a heterologous nucleic acid also includes a nucleic acid that is native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, present in a locus within the genome, expressed from an autonomously replicating vector, linked to a non-native promoter, linked to a mutated promoter, or linked to an enhancer sequence, etc.).
  • Heterologous nucleic acids may comprise plant gene sequences that comprise cDNA forms of a plant gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript).
  • heterologous nucleic acids are distinguished from endogenous plant genes in that the heterologous nucleic acids can be joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the nucleic acid.
  • the heterologous nucleic acids are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
  • nucleic acids in the context of two or more nucleic acids, or two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same (e.g., 75% identity, 80% identity, 85% identity, 90% identity, 95% identity, 97% identity, 98% identity, 99% identity, or 100% identity in pairwise comparison).
  • Sequence identity can be determined by comparison and/or alignment of sequences for maximum correspondence over a comparison window, or over a designated region as measured using a sequence comparison algorithm, or by manual alignment and visual inspection.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity.
  • a "reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence.
  • nucleic acid refers to any RNA or DNA, where the manipulation of which may be deemed desirable for any reason (e.g., treat or reduce the incidence of disease, confer improved qualities, etc.), by one of ordinary skill in the art.
  • nucleic acids include, but are not limited to, coding sequences of structural genes (e.g., disease resistance genes, reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and noncoding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).
  • the term "plant” is used in its broadest sense. It includes, but is not limited to, any species of grass (fodder, ornamental or decorative), crop or cereal, fodder or forage, fruit or vegetable, fruit plant or vegetable plant, herb plant, woody plant, flower plant or tree. It is not meant to limit a plant to any particular structure. It also refers to a unicellular plant (e.g. microalga) and a plurality of plant cells that are largely differentiated into a colony (e.g. volvox) or a structure that is present at any stage of a plant's development.
  • Such structures include, but are not limited to, a seed, a tiller, a sprig, a stolen, a plug, a rhizome, a shoot, a stem, a leaf, a flower petal, a fruit, et cetera.
  • seed refers to a ripened ovule, consisting of the embryo and a casing.
  • Vegetative tissues or vegetative plant parts do not include plant seeds, and instead include non-seed tissues or parts of a plant.
  • the vegetative tissues can include reproductive tissues of a plant, but not the mature seeds.
  • wild-type when made in reference to a gene refers to a functional gene common throughout an outbred population.
  • wild-type when made in reference to a gene product refers to a functional gene product common throughout an outbred population.
  • a functional wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "normal” or "wild-type” form of the gene.
  • the Columbia accession (Col-0) of A. thaliana was used as wild type for all experiments. Plants with jazD were constructed by crossing jazQ ( Campos et al., Nat. Commun. 7: 12570 (2016 )) to other transfer DNA (T-DNA) or transposon insertion mutants obtained from the Arabidopsis Biological Research Center (ABRC; Ohio State University). The following jaz-single mutants were combined with jazQ as described in FIG. 1A-1D , and were named as follows: jaz2-3 (RIKEN_13-5433-1) ( Gimenez-Ibanez et al.
  • these jazD mutations eliminate transcription from Jazl, Jaz2, Jaz3, Jaz4, Jaz5, Jaz6, Jaz7, Jaz9, Jaz10 and Jaz13 genes. Although an amplicon appears in the Jaz4 gel, this amplicon is unrelated to Jaz4 and does not indicate that a Jaz4 transcript was expressed.
  • sieved seeds Seeds retained after sieving (referred to as "sieved seeds") were dried for two weeks in 1.5 mL Eppendorf tubes containing Drierite desiccant. PCR analysis PCR-based genotyping of jazD and lower-order mutants was performed using primer sets flanking DNA insertion sites and a third primer recognizing the T-DNA border (Table 4). Table 4.
  • PCR reactions were performed with the following condition: 95 °C for 5 min, followed by 35 cycles of denaturation (30 s at 95 °C), annealing (30 s at 56 °C) and elongation (1.5 min at 72 °C). Final elongation step was performed at 72 °C for 10 min and completed reactions were maintained at 12 °C.
  • the jaz8 -V mutant was distinguished from wild-type JAZ8 amplicons by digestion with Afl II (New England Biolabs). The presence or absence of full-length JAZ transcripts in Col-0, jazQ, and jazD plants was determined by reverse transcription (RT) PCR.
  • RNA was extracted from rosette leaves of soil-grown plants using a RNeasy kit (Qiagen).
  • cDNA was reverse transcribed with a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, ABI). RT-PCR reactions were performed with primer sets designed to amplify target JAZ genes and the internal control ACTIN1 (At2g37620) by GoTaq Green Master Mix (Promega). Primer sets and additional details of the RT-PCR procedures are provided in Table 5.
  • RGR relative growth rate
  • Seeds were surface sterilized with 50% (v/v) bleach for three min, washed 10 times with sterile water and stratified in dark at 4 °C for two days. Seedlings were grown on 0.7% (w/v) agar media containing half-strength Linsmaier and Skoog (LS; Caisson Labs) salts supplemented with 0.8% (w/v) sucrose and the indicated concentration of MeJA (Sigma-Aldrich). Each square Petri plate (Fisher; 100 x 100 x 15 mm) contained five seedlings per genotype.
  • the eighth true leaf of 40-day-old plants grown under 12-hour-light/12-hour-dark conditions were spotted with 5 ⁇ L of sterile water (mock) or a solution containing 50 ⁇ M coronatine (Sigma-Aldrich, C8115) prepared in sterile water. Photographs were taken two and four days after treatment.
  • Insect feeding assays were performed at 20 °C under a short-day photoperiod of 8-hour light and 16-hour dark. Neonate Trichoplusia ni larvae (Benzon Research) were transferred to fully expanded rosette leaves of 9-week-old plants. Four larvae were reared on each of 12 plants for approximately 12 days, after which larval weights were measured ( Herde et al. Methods Mol Biol 1011:51-61 (2013 )). Botrytis cinerea bioassays were performed as described previously ( Rowe et al. Mol Plant Microbe Interact 20:1126-1137 (2007 )), with minor modifications.
  • Germination assays were performed on half-strength LS agar plates without sucrose. Unsieved seeds were surface sterilized and stratified in dark at 4 °C for two days. Plates were incubated vertically under continuous light at 21 °C and germination was scored daily for seven days by radicle emergence from the seed coat ( Dekkers et al., Planta 218:579-588 (2004 )).
  • Raw sequencing reads were filtered with Illumina quality control tool FASTX-Toolkit (see website at hannonlab.cshl.edu/fastx_toolkit/) and then mapped to TAIR10 gene models by RSEM (version 1.2.25) ( Li et al., BMC Bioinformatics 12:323 (2011 )).
  • mRNA abundances for all Arabidopsis genes were expressed as transcripts per million (TPM). The average TPM ⁇ s.e.m for all genes is shown in Dataset S1, sheet a .
  • DESeq2 version 3.3 ( Anders, Genome Biol 11:R106 (2010 ) was used to normalize expected counts from RSEM and to determine differential gene expression by comparing normalized counts in Col-0 to those in mutants.
  • DAVID version 6.8 ( Huang et al., Nat Protoc 4:44-57 (2009 )) and MapMan (version 3.6.0) ( Thimm et al., The Plant 37:914-939 (2004 )) was used to perform gene ontology (GO) analysis of enriched functional categories. Over-represented and under-represented GO categories among differentially expressed genes were assessed by hypergeometric test with Benjamini & Hochberg's false discovery rate (FDR) correction at P ⁇ 0.05.
  • FDR Benjamini & Hochberg's false discovery rate
  • RNA sequencing data from this study have been deposited in the Gene Expression Omnibus (GEO) database, see website at ncbi.nlm.nih.gov/geo (accession no. GSE116681).
  • GEO Gene Expression Omnibus
  • Proteins extracted from leaf tissue of 23-day-old soil-grown Col-0 and jazD plants were extracted with the following extraction buffer: 100 mM Tris-HCl (pH 6.8), 150 mM NaCl, 10% glycerol (v/v), 4% SDS (w/v), 200 mM DTT, and protease inhibitor (Sigma-Aldrich, 1 tablet/10 mL buffer). Protein concentrations were determined by Bradford assay.
  • Trypsin-digested peptides derived from these proteins were derivatized with a tandem mass tag (TMT) labeling kit (ThermoFisher) for quantification by mass spectrometry (MS) performed at the Michigan State University Proteomics Core Facility (see website at rtsf.natsci.msu.edu/proteomics/). Briefly, protein samples were digested with trypsin using the Filter-Aided Sample Preparation (FASP) protocol according to Wisniewski et al. (Nat Methods 6:359-362 (2009 )).
  • FASP Filter-Aided Sample Preparation
  • Leaf tissue was freeze-dried and used for the measurement of the ratio of 13 CO 2 to 12 CO 2 by mass spectrometry at the Stable Isotope Ratio Facility for Environmental Research, University of Utah (Salt Lake City, UT). Isotopic ratios and CO 2 partial pressure at Rubisco were calculated as described ( Weraduwage et al. Front Plant Sci 6:167 (2015 ); Farquhar et al. Funct Plant Biol 9:121-137 (1982 ); Farquhar et al. Annu Rev Plant Biol 40:503-537 (1989 )).
  • Leaf tissue was harvested from 23-day-old plants grown under our standard long-day conditions. Excised shoots were lyophilized to determine the dry weight. Total protein was extracted using a Plant Total Protein Extraction Kit (PE0230, Sigma-Aldrich) and quantified by Bradford assay. Lipid extraction, thin-layer chromatography (TLC) of polar and neutral lipids, transesterification, and gas chromatography were performed as described previously ( Wang & Benning, J Vis Exp 49:2518 (2011 ); Wang et al. Plant Cell (2018 )).
  • TLC thin-layer chromatography
  • lipid separation was performed by activated ammonium sulfate-impregnated silica gel TLC plates (TLC Silica gel 60, EMD Chemical) with a solvent consisting of acetone, toluene, and water (91:30:7.5 by volume). Lipids were visualized by brief exposure to iodine vapor on TLC plates. Acyl groups of the isolated lipids were then converted to methyl esters, which were subsequently quantified by a gas chromatography. Cell wall was extracted with a solution containing 70% ethanol, chloroform/methanol solution (1:1 v/v) and acetone as described ( Foster et al. J Vis Exp 37:1837 (2010 )). Starch was removed from the extracts using amylase and pullulanase (Sigma-Aldrich). Protein, lipid and cell wall content was normalized to leaf dry weight.
  • Plates were placed in the dark at 4 °C for four days and then incubated horizontally (for leaf biomass) or vertically (for root growth) in growth chambers maintained at 21 °C under 16 h at a light intensity of 80 ⁇ E m -2 s -1 and 8-hour dark. ImageJ was used to measure root length after 11 days. Plant biomass and projected leaf area were measured after 16 days.
  • Example 2 Reduced Growth and Fertility of a jazD Mutant Is Associated with Extreme Sensitivity to JA.
  • This Example describes the growth and fertility of the jazD mutant plants.
  • FIG. 1 The insertion mutations used to construct a series of higher-order jaz mutants are shown in FIG. 1 with which to interrogate the biological consequences of chronic JAZ deficiency in Arabidopsis.
  • the 13-member JAZ family in Arabidopsis is comprised of five phylogenetic groups (I-V) that are common to angiosperms ( FIG. 1B ).
  • the jazQ mutant harbors mutations in the sole member ( JAZ10 ) of group III, all three members of group V ( JAZ3, JAZ4, JAZ9 ), and one member ( JAZ1 ) of the largest group I clade.
  • jazD homozygous jaz1-jaz7,jaz9,jaz10,jaz13 decuple mutant
  • the relative growth rate (RGR) of jazQ was comparable to wild type, despite the reduced biomass of jazQ rosettes at later times in development, which may reflect growth changes occurring before the first time point of sampling (11 days after sowing) or the lack of statistical power needed to resolve small differences in RGR that are compounded over time into larger differences in rosette size.
  • RGR relative growth rate
  • bulk protein, lipid, and cell wall content of rosette leaves were similar between all three genotypes under the growth conditions employed, the ratio of leaf dry weight (DW) to fresh weight was increased in jazD relative to wild type and jazQ.
  • jazD roots and leaves were associated with changes in flowering time under long-day growth conditions.
  • the jazD plants were delayed in their time-to-flowering compared with jazQ but contained a comparable number of leaves at the time of bolting.
  • COR coronatine
  • Wild type and jazQ leaves exhibited visible accumulation of anthocyanin pigments at the site of COR application (i.e., midvein) within 4 days of the treatment, with no apparent signs of chlorosis ( FIG. 2B ).
  • jazD leaves exhibited visible chlorosis at the site of COR application within 2 days of treatment and, strikingly, near complete loss of chlorophyll and spreading of necrosis-like symptoms throughout the leaf 4 days after treatment, leading to tissue death ( FIG. 2B ).
  • silique per plant ⁇ WT 608.3 ⁇ 103.8 21.6 ⁇ 1.3 1.59 ⁇ 0.07 63 ⁇ 11 451 ⁇ 77 jazQ 524.3 ⁇ 98.5 17.3 ⁇ 0.9* 1.70 ⁇ 0.06 58 ⁇ 6 533 ⁇ 100 jazD 192.7 ⁇ 70.0* 16.6 ⁇ 0.7* 1.45 ⁇ 0.08* 37 ⁇ 4* 329 ⁇ 119*
  • Data show the mean ⁇ SD of at least 10 plants per genotype. Asterisks denote significant difference compared with WT plants according to Tukey's HSD test (* P ⁇ 0.05).
  • ⁇ Seed yield was determined by collecting all seeds from individual WT Col-0 and jaz mutant plants. ⁇ Average seed mass was determined by weighing batches of 200 seeds. ⁇ Fully elongated 7th, 9th, and 11th siliques were collected for measurements of silique traits. These traits were used to calculate the estimated number of siliques per plant.
  • the reduced fecundity of jazD resulted from a combination of decreased average mass per seed and lower total seed number per plant. Mutant plants produced fewer seeds per silique, and the size and number of siliques per plant were reduced as well (Table 4). The reduced size of jazD seeds correlated with a reduction in total fatty acid per seed ( FIG. 2D ). Analysis of seed fatty acid profiles showed that jazQ and jazD seeds contain less oleic acid (18:1) and more linoleic acid (18:2), indicating that alterations in fatty acid metabolism occur in these jaz mutants during seed development.
  • Example 3 Constitutive Activation of JA-Mediated and Ethylene-Mediated Defense Pathways in jazD Plants
  • RNA-seq Messenger RNA sequencing
  • RNA-seq data also revealed that ethylene-response genes were highly expressed in jazD but not jazQ.
  • antifungal defense genes controlled by the synergistic action of JA and ethylene were modestly repressed in jazQ but induced in jazD ( FIG. 3C ).
  • genes encoding the AP2/ERFs ERF1 and ORA59 which integrate JA and ethylene signals to promote the expression of antimicrobial compounds, including various defensins (PDFs), pathogenesis-related (PR) proteins, and hydroxycinnamic acid amides (HCAAs) ( FIG. 3C ).
  • PDFs various defensins
  • PR pathogenesis-related proteins
  • HCAAs hydroxycinnamic acid amides
  • jazQ plants were slightly more susceptible than wild type to the necrotrophic pathogen Botrytis cinerea, whereas jazD leaves were more resistant to the spread of disease lesions ( FIG. 3D-3D ).
  • jazQ and jazD differentially affect other ethylene responses.
  • the inventors assessed apical hook formation in ethylene-elicited seedlings. Consistent with studies showing that apical hook formation is attenuated by JA signaling ( Song et al. Plant Cell 26:263-279 (2014 )), FIG.
  • 3F shows that stimulation of hook curvature in response to treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was reduced in jazD but not jazQ seedlings.
  • RNA-seq results and gain additional insight how jazD promotes leaf defense
  • the inventors used quantitative tandem mass spectrometry to quantify global changes in protein abundance in jazD leaves vs. wild type leaves.
  • 149 accumulated to higher in jazD leaves while 120 proteins accumulated to lower levels in jazD leaves (threshold fold-change >1.2, P ⁇ 0.05).
  • GO analysis of the 120 down-regulated proteins revealed enrichment of functional categories related to cytokinin response, cold response, and various functional domains of photosynthesis (Table 5A-5B).
  • Table 7A-7B list biological processes in which proteins whose abundance in jazD leaves was increased or decreased in comparison to wild-type Col-0 based on gene ontology (GO) analysis. Enriched functional categories were determined with DAVID (version 6.8) using the hypergeometric test with Benjamini & Hochberg's false discovery rate (FDR) correction.
  • DAVID version 6.8
  • FDR Benjamini & Hochberg's false discovery rate
  • Table 7A Upregulated in jazD GO ID GO description P value 0009695 jasmonic acid biosynthetic process ⁇ 0.0001 0055114 oxidation-reduction process ⁇ 0.0001 0009611 response to wounding ⁇ 0.0001 0009651 response to salt stress ⁇ 0.0001 0009753 response to jasmonic acid ⁇ 0.0001 0008652 cellular amino acid biosynthetic process ⁇ 0.0001 0000162 tryptophan biosynthetic process ⁇ 0.0001 0050832 defense response to fungus ⁇ 0.0001 0006952 defense response 0.0002 0019762 glucosinolate catabolic process 0.0010 0006564 serine biosynthetic process 0.0113 0080027 response to herbivore 0.0226 0009414 response to water deprivation 0.0336
  • Table 7B Downregulated in jazD GO ID GO description P value 0009735 response to cytokinin ⁇ 0.0001 0015979 photosynthesis ⁇ 0.0001 000
  • Leaves from jazD plants exhibited high expression levels of an agmatine coumaroyl transferase (At5g61160) and an associated transporter (At3g23550) involved in the production of antifungal HCAAs.
  • Transcripts encoding the acyl-CoA N-acyltransferase NATA1 (At2g39030), which catalyzes the formation of the defense compound N( ⁇ )-acetylornithine were 50-fold higher in jazD leaves compared with leaves from wild type and jazQ plants. Such expression was accompanied by increased NATA1 protein abundance.
  • RNA-seq and proteomics data were used to infer metabolic pathways that are altered in jazD leaves. Mapping of differentially expressed genes to Kyoto Encyclopedia of Genes and Genomes pathway databases showed that the tricarboxylic acid (TCA) cycle, oxidative pentose phosphate pathway, sulfur assimilation and metabolism, and various amino acid biosynthetic pathways were among the processes most highly induced in jazD, whereas photosynthesis components were generally down-regulated ( FIG. 4A ).
  • TCA tricarboxylic acid
  • Trp biosynthetic enzymes involved in the production of indole glucosinolates showed particularly high expression at the mRNA and protein levels ( FIG. 4B ).
  • genes encoding enzymes in the phosphoserine pathway that supplies Ser for the biosynthesis of Trp and Cys were highly up-regulated in jazD, as was the abundance of the corresponding enzymes as determined from proteomics data ( FIG. 4B ).
  • Example 4 jazD Plants Exhibit Symptoms of Carbon Starvation.
  • genes involved in starch and sucrose metabolism were generally down-regulated in jazD, including the mRNA and protein abundance of the plastidic starch biosynthetic enzyme phosphoglucomutase (PGM1, At5g51820).
  • RNA-seq data was used to query the expression of genes that are induced by conditions (e.g., prolonged darkness) leading to carbon starvation.
  • SSM sugar starvation marker
  • SSM sugar starvation marker
  • GDH EIN3-regulated glutamate dehydrogenases
  • FIG. 5D-5E show that although exogenous sucrose promotes increased biomass in all genotypes tested, the stimulatory effect on the growth of jazD shoots was statistically greater than that of wild type and jazQ. Exogenous sucrose also enhanced the root growth of jazD in comparison with wild type and jazQ ( FIG. 5F ). Control experiments with sorbitol showed that the growth-promoting effect of sucrose was not attributed to changes in osmotic strength of the growth medium. These data provide evidence that the reduced growth of jazD but not jazQ results in part from a limitation in carbon supply.
  • Example 5 A jaz1-jaz10 and jazl3 Undecuple Mutant Produces Few Viable Seeds
  • JAZ8 The ability of jazD plants to perceive and respond to exogenous jasmonate (JA) suggested that the remaining JAZ proteins in the mutant can actively repress JA-responsive genes.
  • JAZ8 focused on JAZ8 because of its established role in repressing JA responses and the availability of a naturally occurring jaz8-null allele ( Thireault et al. Plant J 82:669-679 (2015 )).
  • the increased expression of JAZ8 in jazD leaves (>15-fold relative to WT) was also consistent with a role in negative-feedback control of JA responses.
  • jazU plants exhibited near complete loss of viable seed production ( FIG. 6C ). Less than 3% of jazU flowers set fruit; although jazU pollen was viable in crosses, among flowers that produced fruit, most senesced and aborted during silique filling. Among the few jazU flowers that did produce seeds, seed set per silique was severely reduced, with recovery of only a few viable seeds per plant. The collective seed-yield phenotype of jazQ, jazD, and jazU supports a key role for JAZ proteins in promoting reproductive vigor.
  • the inventors used jazD in a genetic suppressor screen to identify 11 independent sjd ( suppressor of jazD ) mutants in which rosette growth was partially restored while maintaining enhanced production of defense compounds.
  • CDK8 CYLIN-DEPENDENT KINASE 8
  • At5G63610 CYLIN-DEPENDENT KINASE 8
  • the cdk8 mutation not only partially restores vegetative growth but also fully recovers the low seed yield of jazD, while maintaining robust defense against insect herbivores ( FIG. 7A-7C ).
  • sjd56 -like F2 plants were generated from a cross between sjd56 and jazD parental lines. Sanger sequencing was performed on the genomes of the F2 progeny, demonstrating that each of the fifteen sjd56 -like F2 plants had the C1684T mutation, shown in the nucleic acid segment provided below (SEQ ID NO:121).
  • the sjd56 C1684T mutation truncates the CDK8 protein by altering a glutamine residue to a stop a codon.
  • jazD jaz1-SM, jaz2-RK, jaz3-GK, jaz4-1, jaz5-1, jaz6-DT, jaz7-1, jaz9-4, jaz10-1, jaz13-1 .
  • the progeny of this screen were screened by PCR-genotyping using primer sets flanking DNA insertion sites and a third primer flanking the T-DNA border.
  • This Example illustrates that jazD plants with a null CDK8 mutation (e.g., sjd56 plants) exhibit increased growth and improved resistance to insects compared to jazD and wild type plants.
  • a null CDK8 mutation e.g., sjd56 plants
  • Wild type Col-0 (WT), jazD and sjd56 plants were grown under different conditions.
  • the different plant types were grown under short-day (8-h-light/16-h-dark) conditions, and at 58 days of growth, the rosette fresh weight and projected leaf area of the different plant types was measured.
  • the sjd56 plants exhibit greater rosette fresh weight and greater projected leaf area than the jazD plants, but somewhat less rosette fresh weight and less projected leaf area than wild type plants.
  • WT wild type Col-0
  • jazD jazD
  • sjd56 plants were grown under long-day (16-h-light/8-h-dark) conditions, and at 23 days of growth anthocyanin levels were measured in the leaves of the different plant types.
  • FIG. 8C shows that the anthocyanin levels in leaves of sjd56 plants are significantly greater than in leaves of wild type Col-0 (WT) and jazD plants.
  • FIG. 8D shows the weight of Trichoplusia ni ( T. ni ) after feeding for ten days. As illustrated, substantially more Trichoplusia ni ( T. ni ) were present on wild type Col-0 (WT) and even on jazD plants than on the sjd56 plants.
  • Example 8 cdk8 mutations restore growth and reproduction while delaying vegetative and reproductive transitions of jazD
  • Col-0 (WT) cdk8-1, cdk8-2, jazD, jazD cdk8-1 and jazD cdk8-2 plants were grown under short-day conditions (8-h-ligh/16-h-dark) for 58 days, and the rosette fresh weights and leaf diameters were then measured.
  • FIG. 9A and 9G the rosette fresh weights of jazD cdk8-1 and jazD cdk8-2 plants after 58 days of growth were significantly greater than the rosette fresh weights of jazD plants, approaching the rosette fresh weights of wild type plants.
  • FIG. 9F graphically illustrates that loss of cdk8 increases leaf diameter in jazD plants.
  • FIG. 9B graphically illustrates that as compared to wild type or jazD plants, the time until the first flowers appear was slightly longer for plants with cdk8 null mutations, including the jazD cdk8-1 and jazD cdk8-2 plants.
  • FIG. 9C shows the number of rosette leaves at the time of bolting is greater for cdk8-1, cdk8-2, jazD cdk8-1 and jazD cdk8-2 plants compared to wild type and jazD plants.
  • Seed yield and seed mass of WT, cdk8-1, cdk8-2, jazD, jazD cdk8-1 and jazD cdk8-2 plants were also measured. Seed numbers were evaluated by collecting all seeds from individual plants. Average seed mass was determined by weighing batches of 100 seeds.
  • seed yield and seed mass for plants with cdk8 null mutations, including the jazD cdk8-1 and jazD cdk8-2 plants was greater than determined for jazD plants, and was similar to that observed for wild type plants.
  • Example 9 cdk8 mutations partially recover the defense phenotypes of jazD
  • This Example illustrates the pest resistance provided by combining cdk8 null alleles into jazD plants.
  • FIG. 10A provides images of larvae isolated from the different plant types. As illustrated, larval sizes are significantly smaller when maintained on jazD, jazD cdk8-1 and jazD cdk8-2 plants than larvae maintained on wild type plants.
  • FIG. 10B graphically illustrates the weights of larvae isolated from the different plant types. The data show the mean ⁇ SD of at least 18 larvae per genotype. As shown in FIG. 10B , larval weights are significantly less when the larvae feed on jazD, jazD cdk8-1 and jazD cdk8-2 plants.
  • Example 10 The increased production of defense compounds in jazD is partially regulated by CDK8.
  • This Example illustrates production of various plant defense compounds by jazD and jazD cdk8 plants.
  • Col-0 (WT), cdk8-1, jazD, and jazD cdk8-1 plants were grown under long-day conditions (16-h-light/8-h-dark) in soil.
  • Defense compounds were extracted from leaves of 23-day-old plants grown under long-day conditions (16-h-light/8-h-dark).
  • FIG. 11A graphically illustrates anthocyanin levels in leaves of 25-day-old wild type Col-0 (WT), cdk8, jazD and jazD cdk8 plants.
  • FIG. 11B-11D graphically illustrate indole glucosinolates, N ⁇ -acetylornithine, and hydroxycinnamic acid amides (HCAAs) levels in WT, cdk8, jazD and jazD cdk8 leaves. Comparison of Peak area for the indicated compound in the WT sample was set to "1" and the peak area of the same compound in other genotypes was normalized to the WT sample.
  • I3M indol-3-ylmethyl, glucobrassicin
  • OH-I3M 4-hydroxyindol-3-ylmethyl, hydroxyglucobrassicin
  • 4MOI3M 4-methoxyindol-3-ylmethyl, methoxyglucobrassicin
  • 1MOI3M 1-methoxyindol-3-ylmethyl, neoglucobrassicin.
  • Data show the mean ⁇ SD of three biological replicates per genotype. Letters denote significant differences according to Tukey's HSD test (P ⁇ 0.05).
  • Col-0 WT
  • cdk8-1 cdk8-1, jazD, and jazD cdk8-1 plants were grown under long-day conditions (16-h-light/8-h-dark) in soil and leaves of 25-day-old were collected for quantitative PCR analysis.
  • FIG. 11E graphically illustrates relative expression levels of VEGETATIVE STORAGE PROTEIN 2 (VSP2, AT5G24770) while FIG. 11F graphically illustrates relative expression levels of PLANT DEFENSIN 1.2 (PDF1.2, AT5G44420).
  • PP2A AT1g13320 was used for qPCR normalization. Data show the mean ⁇ SD of three biological replicates per genotype. Letters denote significant differences according to Tukey's HSD test (P ⁇ 0.05).
  • Example 11 cdk8 mutations promotes production of aliphatic glucosinolates in jazD
  • This Example illustrates some of the compounds generated by leaves of plants of various genotypes, including the from leaves of jazD, cdk8, jazD and jazD cdk8 plants.
  • FIG. 12 graphically illustrates aliphatic glucosinolate levels in WT, cdk8, jazD and jazD cdk8 leaves.
  • Example 12 Increased resistance of jazD to 5-methyl-tryptophan (5-MT) is partially dependent on CDK8
  • FIG. 13A is a schematic of tryptophan biosynthesis from chorismate. Tryptophan feedback inhibits the activity of anthranilate synthase (AS). Although 5-methyl-tryptophan (5-MT) inhibits anthranilate synthase activity, it cannot be used for the production of proteins.
  • the abbreviations used in FIG. 13A are: TRP, anthranilate phosphoribosyltransferase; PAI, phosphoribosylanthranilate isomerase; IGPS, indole-3-glycerol-phosphate synthase; TSA, tryptophan synthase alpha subunit; TSB, tryptophan synthase beta subunit.
  • FIG. 13B graphically illustrates root length of WT, cdk8-1, jazD, and jazD cdk8-1 10-day-old seedlings grown on medium supplemented with 0 or 15 ⁇ M of 5-methyl-tryptophan (5-MT).
  • the data shown in FIG. 13B are the mean ⁇ SD of at least 24 seedlings per genotype at each 5-MT concentration, while the letters denote significant differences according to Tukey's HSD test (P ⁇ 0.05).

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